Development of strains has been continuously conducted to enhance price competitiveness of methods of producing metabolites using microorganisms. A conventional and effective combinational approach for developing new strains includes producing a strain library based on a producing strain and screening strains having improved characteristics from the strain library. For example, the combinational approach includes a case of development of a monosodium glutamate (MSG)-producing strain using a production method converted from an extraction method of the 1960s to a fermentation method using microorganisms.
To produce the strain library, UV irradiation, methods such as chemical mutagenesis including adding a chemical mutagen such as nitrosoguanidine (NTG), and the like were used in the past. In modern times in which biomolecular tools and biochemical knowledge have been accumulated, various methods of producing a strain library, such as mutagenesis of genes based on a polymerase chain reaction (PCR), genome shuffling using protoplast fusion, and random insertion using a transposon have been proposed. A strain library may be produced using the diverse methods of producing a strain library described above since the probability of the strain library including the high target-metabolite-producing microorganisms increases with an increase in size of the strain library.
Various analysis methods have been used to determine the productivity of a target metabolite in a strain. Liquid/gas chromatography (LC/GC) is a method of culturing individual strains and analyzing concentrations of metabolites in a culture broth and the strain. Such a method is available for quantitative analysis when it can be used to detect most of the metabolites and obtain a standard assay curve. However, the method can be used to analyze only one mutant strain at a time, and thus is inefficient for analysis of a library of strains greater than a predetermined size due to low throughput.
A method of analyzing a metabolite using multiplates is a method of analyzing a change in concentration of the metabolite in a sample, which includes putting a mutant strain into partitioned wells and measuring a change in chromogenesis, optical density or fluorescence intensity of a small amount of the sample. Since a small amount of the sample and the multiplates are used, relatively many mutant strains can be analyzed at the same time. However, the method has a problem in that its throughput capacity is insufficient to analyze a library of strains having a large size prepared by the production method. Also, the production method has a narrow application since a chromogenic reaction can be performed using the metabolite as a substrate, or it is applicable to metabolites in which a change in optical density or fluorescence intensity can be measured.
A method of screening a producing strain using a genetic biosensor is used to immediately convert a concentration of a synthesized target metabolite into a detectable signal and detect the detectable signal. When a biosensor specific to a target metabolite is developed and used, a proper detector may be employed to observe a change in concentration of the target metabolite which cannot be detected visually.
A fluorescence-activated cell sorting (FACS) technique is used to detect the fluorescence emitted from individual strains while allowing mutant strains to flow through a detector. Such a technique has a throughput capacity of more than 109 cells since the fluorescence may be detected quickly while allowing a large amount of cells to flow at the same time. When the target metabolite emits the fluorescence, a large library can be analyzed in a relatively quick and easy manner. Thus, it is possible to efficiently screen the high target metabolite-producing microorganism. However, such a technique has a problem in that it is applicable only to metabolites emitting fluorescence.
Finally, a selection method may be used. Such a method is technology designed such that only the strains producing a high concentration of a target metabolite in the strain library survive. Such a method has a very high throughput, and thus may be used to effectively screen only the high target metabolite-producing microorganisms from a library of strains having large sizes. However, such technology can be applied only when the concentration of the target metabolite is associated with the growth or survival of the strains.
Meanwhile, a riboswitch is a biosensor for sensing a concentration of a certain metabolite in cells and regulating expression levels of genes positioned downstream from the riboswitch, and has very high specificity and affinity to substrates. Also, techniques of producing aptamers binding to a certain metabolite using a systematic evolution of ligand by exponential enrichment (SELEX) technique, and constructing riboswitches based on the aptamers have been developed. Therefore, it is possible to develop a riboswitch capable of specifically and sensitively sensing only a metabolite which the present applicant wish to screen and regulating expression levels of genes positioned downstream from the riboswitch.
When a selectable marker gene is inserted downstream from the riboswitch developed thus, it is possible to obtain an RNA device capable of regulating the expression of the selectable marker gene according to the concentration of a target metabolite. When the RNA device is introduced into the strain libraries produced using the various methods, the expression level of the selectable marker gene varies according to the concentration of the target metabolite in each strain. In this case, when a strain candidate transformed with an artificial selection circuit is exposed to a suitable selective pressure to adapt to the selectable marker gene of the RNA device, only the strains producing a high concentration of the target metabolite will survive.